3-deazaguanine

ABSTRACT

3-Deazaguanine and physiologically compatible salts thereof, useful as a broad spectrum anti-viral agent, are disclosed.

United States Patent [191 Rousseau et al.

5 1 S-DEAZAGUANINE [75] Inventors: Robert J. Rousseau, Laguna Niguel;Roland K. Robins, Santa Ana, both of Calif.

{73] Assignee: ICN Pharmaceuticals, Inc., Irvine.

Calif.

[22] Filed: July 6, 1973 [2l] Appl. No.: 377,078

[52] US. Cl. 6. 260/296 H; 424/263; 260/309 {51] Int. Cl C07d 31/42 [58]Field of Search 260/296 H [56] References Cited OTHER PUBLICATIONSKorte, Chem Ber. Vol 85, pages l0l2 to 1022 (1952).

[ July 22, 1975 Primary E.mminer-.l0hn D. Randolph Auorney. Agent, orFirm-Lyon & Lyon [57] ABSTRACT 3-Dcazaguanine and physiologicallycompatible salts thereof, useful as a broad spectrum anti-viral agenl,are disclosed.

4 Claims, No Drawings 1 3-DEAZAGUANINE BACKGROUND OF THE INVENTIONDuring the past decade. many nucleoside analogs have found to exhibitgood antitumor and antiviral activities. Among the presently knownsynthetic nucleosidic antiviral agents. the more important generally areconsidered to be S-iodo-2'-deoxyuridine (IDU), 9-B-D-arabinofuranosyladenine (ara-A) and l-B-D- arabinofuranosylcytosine(ara-C). These compounds. however, are only active against a limitedspectrum of viruses which does not include those causing respiratorydiseases in man (influenza, common cold). The only nucleosidic analog ofwhich are aware that is active against these respiratory disease virusesis l-B-D- ribofuranosyl-l,2.4-triazole-3-carboxamide which is describedin copending United States patent application, Ser. No. 240,252, filedMar. 3|. I972, entitled l,2,4-Triazole Nucleosides, now U.S. Pat.

SUMMARY OF THE INVENTION The present invention thus relates to3-deazaguanine and physiologically compatible salts as represented bythe following:

XN N YN N H in which Y is H and X is H or an alkalai metal.

DETAILED DESCRIPTION OF THE INVENTION The compounds of this inventionmay be prepared in accordance with the following schematicrepresentation, as set forth in the illustrative esamples which follow',in which temperatures and melting points are ex- No. pressed in degreescentigrade.

-. N It" m 1956-01 D D n rs x J -t ,1 -i w 0 0 f] i Z a l 0 m N Ami-(W NJ I H N N 2 If n H 3,798,209 granted Mar. I9, 1974, which application isEXAMPLE I a continuation in part of Ser. No. 149,0]7, filed June 1, I971entitled l,2,4-Triazole Nucleosides, now abancloned and assigned to thesame assignee as this application.

Certain derivatives of this latter compound have also been found to havesignificant antiviral activity, as have the triazole bases,l,2,4-triazole-3-carboxamide and l,2,4-triazole-3-thiocarboxamide. Evenwith the advent of such compounds, however, and the discovery of theirantiviral efficacy, there remains a need for compounds which are capableof effectively inhibiting virus infections, especially respiratorydisease viruses.

With the foregoing need in mind, we determined to synthesize thepyridine base, 6- aminoimidazo[4,5clpyridin-4-one (3-deazaguanine), acompound not previously reported, and derivatives thereof to see whethersuch compounds would possess effective antiviral capability.

Methyl 4(5 )-acetamido-2-imidazole-S(4)-carboxylate (Compound 2)Compound I (20.4 g, 0.103 mole) was added to I800 ml of methanolsaturated at 0 with ammonia, and the amber solution which resulted wasstirred for l6 hr at room temperature. The light orange solution wasevaporated in vacuo to dryness, the residue was triturated with 50 ml ofmethanol, and then collected and air dried to yield 2 16.6 g (88%), mp.230-232 (literature m.p. 242244).

R. K. Robins et al.. J. Org. Chem. 28, 3041 (1963).

EXAMPLE [I Methyl 4(5 )-acetonitrile-2-imidazole-5(4 )-carboxylate(Compound 3) 16.6 g, 0.097 mole of compound 2 was refluxed in 800 ml ofphosphorousoxychloride for three hours. The

excess phosphorous oxychloride was removed in vacuo using a rotaryevaporator. and the dark syrupy residue was treated with 100 ml of iceuntil all of it had dissolved (an acetone dry-ice bath was used tomaintain the temperature of the solution between -l0 C throughout thisprocess). The dark solution was carefully neutralized until the pHstabilized at 6 (pHydrion paper) while the temperature was maintainedbelow 15 C. The dark colored mixture was allowed to stand at 4 C for 16hr. the precipitate was collected, washed with ice water (3 X 50 ml).and then air dried to yield 3. 12.4 g (77%. m.p. l68-l70. A small amountof this material was recrystallized from water to provide an analyticalsample. m.p. 170l7l, A,,,.,," 222 nm. 10.700; A 251 nm. 10.600; n.m.r.(DMSO-d 83.89 (s. 3H OCH 84.20 (s. 2H. CH 7.90 (s. 1H. H ).'13.3(br.1H.NH).

Anal. Calcd for C H,O.,N c. 50.91; H. 4.27; N. 25.45. Found: C. 50.70;H. 4.25; N. 25.35.

1 EXAMPLE III 4( 5 )-Acetonitri1e-2-imidazole-5( 4)-carboxamide(Compound 4) Compound 3 (5.0 g. 0,0303 moles) was heated with 200 ml ofliquid ammonia in a steel reaction vessel for 48 hr. The excess ammoniawas allowed to evaporate to dryness, the residue was taken up in ca 20ml of water. collected and air dried to yield 2.85 g (62.5%) of compound4. m.p. 225228. A small amount of this material was recrystallized fromwater to give an analytical sample (beige needles). m.p. 230-232.

Anal. Calcd. for C..-H,.N.O 1 C. 48.00; H. 4.03; N. 37.32. Found: C.48.14; H. 3.97; N. 37.51.

EXAMPLE IV 6-Aminoimidazo[4.5-c ]pyridin-4-one (3-deazaguanine)(Compound 5) Method A. 1.5 g (0.01 mole) of compound 4 was refluxed inml of 10% Na,CO for 4 hr. neutralized to pH 6 with concentratedhydrochloric acid and then allowed to stand at 4 C for 16 hr. Theresulting solid was collected. washed well with ice water and dried 8 hrat 100 in vacuo to yield 750 mg (50%) of compound 5. m.p. 300.

Anal. Calcd for C H ON C. 48.00; H, 4.03; N, 37.32. Found: C. 47.99 H.4.10; N. 37.19.

Method B. Compound 3 (5.0 g. 0.0303 mole) was heated in 200 ml liquidammonia in a steel reaction vessel at 100 C for 96 hr. The ammonia wasallowed to evaporate. and the residue was taken up in 60 ml of H 0.collected and air dried to yield 2.85 g (56%) m.p. 300. This wasrecrystallized from I30 ml of water to afford 2.10 g ofgre enish-brownneedles which had the same characteristics chromatographically andspectrometrically as the product from Method A.

EXAMPLE V b-Aminoimidazo[4.5-c]pyridin4-0ne (3-deazagua nin e) viaNI-LCI catalysis.

A solution of 0.50 g (3 mmole) of compound 3 and 0.50 g NH.CI in 20 mlofmethanol. into which NH; had been bubbled for 2 minutes at ambienttemperature. was enclosed in a steel bomb and heated in an oil bathmaintained at for 16 hours. The contents were evaporated. taken up in 20ml hot H 0, and filtered. Tlc showed 3-deazaguanine as the soledetectable product. Cooling deposited 0.15 g product (33%).

It will be apparent to those skilled in the art, given the foregoingdisclosure. that acid addition salts can be prepared following thestandard procedure known in the' art. as by suspension of 3-deazaguanjnein water and reaction with the appropriate acid reactant such ashydrochloric acid. sulfuric acid. etc. Similarly. the basic salts may beformed by reaction with alkali metalic bases such as sodium hydroxide.etc.

EXAMPLE V] In this example. 3-deazaguanine was tested to determine itsin vitro antiviral efficacy, using the virusinduced cytopathogeniceffect (CPE) method of Sidwell. et al. (Applied Microbiology 22:797-801.1971). Briefly. the CPE procedure includes the dissolution of theantiviral agent in a cell culture medium consisting of vitamins. aminoacids. serum, buffer. penicillin. streptomycin and indicator dye inwater. The virus suspended in the cell culture medium was added to anestablished monolayer of KB cells or RI(- 1 3 cells, and an equal volumeof the antiviral agent was then added within 15 minutes. The infectedtreated cells were graded following microscopic examination. Controlsfor each experiment include cell controls (cells and cell culture mediumonly), virus controls (cells and virus and cell culture medium) andtoxicity controls (cells and chemical and cell culture medium).

The virus rating (VR) system of Sidwell et al.. described in AppliedMicrobiology, supra, was used to evaluate the degree of significance ofCPE inhibition. A VR greater than 0.5 is indicative of significantantiviral activity and a VR of less than 0.5 suggests slight antiviralactivity.

The results of the in vitro testing are shown in Table I which follows,as is comparative data for the compound.1-B-D-ribofuranosyl-l.2,4-triazole-3- carboxamide.

TABLE I IN VITRO ANTIVIRAI. ACTIVITY OF 3-DEAZAGUANINE ANDI-fiD-RlBOFURANOSYL-l.2.4-TRIAZOLE-3-CARBOXAMIDE Virus 1-fiD-Ribofuranosyl- Type A influenza Type 1 Rhino Type 2 Rhino Type SRhino Type 13 Rhino Type 30 Rhino Type 56 Rhino Type 3 ParainfluenzaType 1 Herpes simplex TABLE I-- Continued 1N VITRO ANTlVlRAL ACTIVITY OF3-DEAZAGUAN1NE AND l-B-D-RIBOFURANOSYL-l.2,4-TRlAZOLE-3-CARBOXAMlDECytotoxicity-Chick Embryo-Cells Cytoroxicity expressed as the dosecausing after 72 hr exposure to the compound at no visible changes tothe cells as examined microscopically 37C. KB cells: Humanadenocarcinoma of the nasopharynx:

RK-l 3 cells: Continuous passage rabbit kidney. Chick embryo cells:Primary cells derived from minced 9-day-old chicken embryos.

EXAMPLE Vll 3-Deazaguanine was also tested for effectiveness againstinfluenza A virus in an animal experiment. In this study, 16-17 g.female Swiss Webster mice were lightly anesthetized and infectedintranasally with a 75% lethal dose (LD of strain Japan 305 of influenzaA virus. Fifteen minutes prior to their exposure to virus, the mice weretreated intraperitoneally with 3- deazaguanine or l-B-D-ribofuranosyl-l,2,4-triazole-3- carboxamide, using varying doses of each suspended insterile physiological saline. Virus control mice were pretreated withsaline only, and toxicity control animals were treated with thepertinent dosages of each drug but only sham-infected. The animals wereob served over a 21-day period, with deaths occurring during this periodrecorded daily. The results of this study, summarized in Table 11,indicate both compounds to have a significant anti-influenza viruseffect.

TABLE 11 3-Deazaguanine has also demonstrated preliminary anti-bacterialactivity against E-Coli (gram negative) and staphylococcus aureus (grampositive).

When 3-deazaguanine is prepared synthetically. methyl4(5)-acetamide-2-imidazole-5(4)-carboxylate is reacted with adehydrating agent such as phosphorous oxychloride, oxalyl chloride,thionyl chloride, etc. under refluxing conditions to yield methyl 4(5)-acetonitrile-Z-imidazole-S( ll-carboxylate, which is then heated withliquid ammonia at a temperature between about 100 and 150C. to give4(5)-acetonitrile- 2-imidazole-5(4)-carboxamide. This compound is thenreacted, under refluxing conditions, with an alkalai metal carbonatesuch as sodium carbonate, to provide 3-deazaguanine. Alternatively.3-deazaguanine may be prepared directly from the acetonitrilecarboxylatc by treatment with ammonium chloride in an aliphatic alcoholsuch as methanol. etc., in the presence of ammo- EFFECT OF A SINGLEINTRAPERITONEAL INJECTION OF 3-DEAZAGUANINE OR 1 -B-D- RIBOFURANOSY L- l,ZATRIAZOLE-Il-CARBOXAM1DE ON INFLUENZA A VlRUS INFECTIONS 1N MlCEToxicity Infected, Treated Dosages Control Survivor Mean SurvivalSurvival Time Drug [mg/kg) Surv/Total Surv"/Total increase p" Time(days) Increase p" S-Deazaguanine 400 0/5 l/lO 9.3 0.05

100 5/5 5/10 0.1 9.0 0.05 l-B-D-Ribofur- 800 5/5 1/10 9.0 0.05anosy1-l.2,4- triazole-Il-car- 400 5/5 2/10 l 1.4 0.01 boxamide 200 5/54/10 0.2 9.3 0.05 Saline 4/20 7.8

"Day 21 survivors.

Probability [chi square analysisl. Animals dying on or before day 21.Probability (t test).

it will be appreciated from Examples V1 and VII that 3-deazaguanine hasshown a broad spectrum of antiviral activity and is particularlyeffective against respiratory disease viruses. In several instances, thecompound demonstrated significantly greater in vitro activity than thecomparative compound, l-B-D-ribofuranosyl- 1,2,4-triazole-3-carboxamide.in view of the close structural similarity to B-deazaguanine and theusual increased solubility of the salts of active pyridine compounds,the acid addition salts would also be expected to exhibit antiviralactivity.

nia at a temperature between about and about C.

We claim:

1. A compound of the structure:

subsequently refluxing such acetonitrile carboxamide in the presence ofan alkalai metal carbonate to afford 3-deazaguanine.

4. The process of claim 3 in which the acetonitrile carboxylate istreated with NH Cl in an aliphatic alcohol and in the presence ofammonia at a temperature of about l00 to about C. to afford 3-deazaguanine.

1. A COMPOUND OF THE STRUCTURE:
 2. 6-Aminoimidazo pyridin-4-one.
 3. Aprocess of preparing 3-deazaguanine comprising reacting methyl4(5)-acetamido-2-imidazole-5(4)-carboxylate under refluxing conditionswith a dehydrating agent to afford methyl4(5)-acetonitrile-2-imidazole-5(4)-carboxylate, heating suchacetonitrile compound to a temperature of about 100 to about 150*C. inthe presence of liquid ammonia to provide4(5)-acetonitrile-2-imidazole-5(4)-carboxamide, and subsequentlyrEfluxing such acetonitrile carboxamide in the presence of an alkalaimetal carbonate to afford 3-deazaguanine.
 4. The process of claim 3 inwhich the acetonitrile carboxylate is treated with NH4Cl in an aliphaticalcohol and in the presence of ammonia at a temperature of about 100* toabout 150*C. to afford 3-deazaguanine.